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抗體的生物素化標(biāo)記實(shí)驗(yàn)要點(diǎn)：

1. 核有絲分裂器NuMA蛋白抗體說(shuō)明書(shū) 如在反應(yīng)混合液中有疊氮鈉或游離氨基存在,會(huì)抑制標(biāo)記反應(yīng)。因此,蛋白質(zhì)在反應(yīng)前要對(duì) 0.1mol/L碳酸氫鈉緩沖液或0.5mol/L硼酸緩沖液充分透析；

2.所用的NHSB及待生物素化蛋白質(zhì)之間的分子比按蛋白質(zhì)表面的ε-氨基的密度會(huì)有所不同,選擇不當(dāng)則影響標(biāo)記的效率,應(yīng)先用幾個(gè)不同的分子比來(lái)篩選最適條件；

3.用NHSB量過(guò)量也是不利的,抗原的結(jié)合位點(diǎn)可能因此被封閉,導(dǎo)致抗體失活；

4.由于抗體的氨基不易接近可能造成生物素化不足,此時(shí)可加入去污劑如 Triton x-100, Tween20等；

5.當(dāng)游離ε-氨基(賴(lài)氨酸殘基的氨基)存在于抗體的抗原結(jié)合位點(diǎn)時(shí),或位于酶的催化位點(diǎn)時(shí),生物素化會(huì)降低或損傷抗體蛋白的結(jié)合力或活性；

6.生物素還可能與不同的功能基團(tuán),如羰基、氨基、巰基、異咪唑基及苯酚基,也可與糖基共價(jià)結(jié)合；

7.交聯(lián)反應(yīng)后,應(yīng)充分透析,否則,殘余的生物素會(huì)對(duì)生物素化抗體與親和素的結(jié)合產(chǎn)生競(jìng)爭(zhēng)作用；

8.在細(xì)胞的熒光標(biāo)記實(shí)驗(yàn)中,中和親和素的本底低,但由于鏈霉親和素含有少量正電荷,故對(duì)某些細(xì)胞可導(dǎo)致高本底。

產(chǎn)品訂購(gòu)信息：
英文名稱(chēng)  Anti-NuMA 

中文名稱(chēng)   核有絲分裂器NuMA蛋白抗體說(shuō)明書(shū) 

別    名  NUMA 1; NuMA protein; NUMA1; SP H antigen; Structural nuclear protein; Centrophilin stabilizes mitotic spindle in mitotic cells; Nuclear mitotic apparatus protein 1; Nuclear mitotic apparatus protein; NUMA1_HUMAN.

濃    度   1mg/1ml

規(guī) 格  0.2ml/200μg

抗體來(lái)源   Rabbit

克隆類(lèi)型   polyclonal

交叉反應(yīng)   Human, Mouse, Rat, Dog 

產(chǎn)品類(lèi)型   一抗  

研究領(lǐng)域     細(xì)胞生物 免疫學(xué) 染色質(zhì)和核信號(hào) 神經(jīng)生物學(xué)

蛋白分子量  predicted molecular weight: 238kDa

性    狀   Lyophilized or Liquid

免 疫 原 KLH conjugated synthetic peptide derived from human NuMA 

亞    型   IgG

純化方法   affinity purified by Protein A

儲(chǔ) 存 液   0.01M PBS, pH 7.4 with 10 mg/ml BSA and 0.1% Sodium azide

核有絲分裂器NuMA蛋白抗體說(shuō)明書(shū) 產(chǎn)品應(yīng)用    ELISA=1:500-1000 IP=1:20-100 IHC-P=1:100-500 IHC-F=1:100-500 IF=1:100-500

（石蠟切片需做抗原修復(fù)） 

 not yet tested in other applications.

 optimal dilutions/concentrations should be determined by the end user.  

保存條件  Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. The lyophilized antibody is stable at room temperature for at least one month and for greater than a year when kept at -20°C. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. 

Important Note  This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications. 

產(chǎn)品介紹 NuMA (Nuclear Mitotic Apparatus Protein) is an intranuclear protein and present in nucleus during interphase. At the onset of mitosis, it redistributes from the nucleus to two centrosomal structures that later will become part of the mitotic spindle pole. After anaphase, the protein redistributes from the spindle polar region into reforming nucleus. NuMA is an essential protein during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly. Recently a study shows that NuMA is cleaved to a 180 to 200kDa during apoptosis.

Function : Highly abundant component of the nuclear matrix where it may serve a non-mitotic structural role, occupies the majority if the nuclear volume. Required for maintenance and establishment of the mitotic spindle poles, functionning as a tether linking bulk microtubules of the spindle to centrosomes. May be involved in coordination of the alignment of the mitotic spindle to the cellular polarity axis, which is a prerequisite for asymmetric cell divisions.Subunit : Homodimer. Also forms multiarm oligomers by association of C-terminal tail domains, oligomers may further assemble to form a hexagonal nuclear lattice-like network. Interacts with TNKS.Subcellular Location : Nucleus matrix. Chromosome. Cytoplasm, cytoskeleton, spindle pole. Note=Resides in the nuclear matrix during interphases. Dissociates from condensing chromosomes during early prophase, and relocates to the spindle poles via dynein/dynamin association, it remain there until the anaphase onset. Before the complete disintegration of the nuclear lamina. As mitosis progresses it reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident.Isoform Numa-m: Cytoplasm. Note=Mainly clustered at the centrosomal region.Isoform Numa-s: Cytoplasm. Note=Mainly clustered at the centrosomal region.Post-translational modifications : ADP-ribosylated by TNKS during mitosis.Phosphorylated in the C-terminal tail during mitosis, probably by CDK1. Phosphorylation increases solubility and promotes association with dynein and subsequent translocation to the spindle poles.Database links : UniProtKB/Swiss-Prot: Q14980.2NuMA (Nuclear mitotic apparatus protein)是細(xì)胞核基質(zhì)中的一種高分子量蛋白質(zhì)，具有一個(gè)特殊的細(xì)胞周期依賴(lài)性.核有絲分裂器蛋白NuMA與人類(lèi)細(xì)胞的惡性增殖相關(guān).

抗體的鑒定：

1）核有絲分裂器NuMA蛋白抗體說(shuō)明書(shū) 抗體的效價(jià)鑒定：不管是用于診斷還是用于,制備抗體的目的都是要求較高效價(jià)。不同的抗原制備的抗體,要求的效價(jià)不一。鑒定效價(jià)的方法很多,包括有試管凝集反應(yīng),瓊脂擴(kuò)散試驗(yàn),酶聯(lián)免疫吸附試驗(yàn)等。常用的抗原所制備的抗體一般都有約成的鑒定效價(jià)的方法,以資比較。如制備抗抗體的效價(jià),一般就采用瓊脂擴(kuò)散試驗(yàn)來(lái)鑒定。

2）抗體的特異性鑒定：抗體的特異性是指與相應(yīng)抗原或近似抗原物質(zhì)的識(shí)別能力。抗體的特異性高,它的識(shí)別能力就強(qiáng)。衡量特異性通常以交叉反應(yīng)率來(lái)表示。交叉反應(yīng)率可用競(jìng)爭(zhēng)抑制試驗(yàn)測(cè)定。以不同濃度抗原和近似抗原分別做競(jìng)爭(zhēng)抑制曲線,計(jì)算各自的結(jié)合率,求出各自在IC50時(shí)的濃度,并按公式計(jì)算交叉反應(yīng)率。 

如果所用抗原濃度IC50濃度為pg/管,而一些近似抗原物質(zhì)的IC50濃度幾乎是無(wú)窮大時(shí),表示這一抗血清與其他抗原物質(zhì)的交叉反應(yīng)率近似為0,即該血清的特異性較好。

3）抗體親和力：是指抗體和抗原結(jié)合的牢固程度。親和力的高低是由抗原分子的大小,抗體分子的結(jié)合位點(diǎn)與抗原決定簇之間立體構(gòu)型的合適度決定的。有助于維持抗原抗體復(fù)合物穩(wěn)定的分子間力有氫鍵,疏水鍵,側(cè)鏈相反電荷基因的庫(kù)侖力,范德華力和空間斥力。親和力常以親和常數(shù)K表示,K的單位是L/mol?？贵w親和力的測(cè)定對(duì)抗體的篩選,確定抗體的用途,驗(yàn)證抗體的均一性等均有重要意義。

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